Tissue- and temporal-specific gene expression and regulation are found, inter alia, during sexual reproduction in eukaryotes. In plant gametogenesis, important cytological and biochemical changes occur during pollen development when the asymmetric mitotic division of the haploid microspore results in the formation of two cells; each with different developmental fates. The vegetative cell supports pollen growth while the generative cell undergoes mitosis and develops into sperm cells. Messenger RNAs specific to both pathways within pollen have been identified in plants such as maize, tomato, tobacco, rice and pansy; and messages specific to pollen or to one or more other cell types within anther such as tapetum, epidermis and stomium have also been identified.
Pollen gene expression during differentiation involves an estimated 24,000 genes (Willing, et al., "An Analysis of the Quantity and Diversity of mRNA's From Pollen and Shoots of Zea mays"; Theor. Appl. Genet.; Vol. 75; pp. 751-753; (1988)), however only 10% of clones from a cDNA library are male-specific (Stinson, et al., "Genes Expressed in the Male Gametophyte and Their Isolation"; Plant Physiol.; Vol. 83; pp. 442-447; (1987)) and the percentage of genes expressed in pollen that are pollen-specific is between 5% and 80% (willing, et al., "An Analysis of the Quantity and Diversity of mRNA's From Pollen and Shoots of Zea mays"; Theor. Appl. Genet.; Vol. 75; pp. 751-753; (1988)). This complex process of microsporogenesis involves the sequential production of many gene products.
To date male-specific genes have been cloned from plants: two of these, the maize Ms45 gene (U.S. Pat. No. 5,478,369) and the Arabidopsis Ms2 gene (Mark, G. M., et al., Nature: Vol. 363; pp. 715-717; (1993)), have been shown to be required for pollen development. Other examples of male-specific promoters in plants include ZM13, PG, and SGB6.
The Zm13 promoter is disclosed in U.S. Pat. No. 5,086,169. It consists of 1315 base pairs and is from a pollen specific gene described by Hanson, et al., Plant Cell; Vol. 1; pp. 173-179; (1989). This gene hybridizes to mRNA found only in pollen.
Another pollen-specific promoter has been isolated and characterized upstream of the pollen-specific polygalacturonase gene (PG) U.S. Pat. No 5,412,085. This promoter region encompasses 2687 base pairs and is expressed predominantly in pollen and emergent tassel, but not in pre-emergent tassel. U.S. Pat. No. 5,545,546, also from Allen, describes another pollen-specific promoter from the maize polygalacturonase gene. It is only expressed in pollen and in emergent tassel.
U.S. Pat. No. 5,470,359 describes a regulatory region from the SGB6 gene of maize which confers tapetum specificity. The tissue of expression, the tapetum, is a layer of cells that surrounds the microsporogenous cells in the anther and provides nutrients thereto.
Nine anther-specific cDNA and genomic clones from tobacco are described in U.S. Pat. No. 5,477,002. The cDNA clones were anther-specific by Northern analysis, yet differed in developmental profiles. Clone Ant32 is tapetal-specific.
European Pat. No. 0 420 819 Al describes the method of producing male sterile plants with the wun1 gene.
PCT WO 90/08825 describes anther-specific cDNAs TA13, TA26 and TA29 and their use in a male sterility system.
PCT WO 90/08825 explains male-sterility genes pMS10, pMS14 and pMS18 and their use with the GUS reporter gene.
U.S. Pat. No. 5,589,610 details a promoter corresponding to anther-specific cDNA and anther-preferred cDNA AC444.
The use of a plant promoter and an exogenous gene to effect a change in the genetic make-up of plants is known in the art (U.S. Pat. Nos. 5,432,068, 5,412,085, 5,545,546, 5,470,359 and 5,478,369) These patents discuss plant expression cassettes with a tissue-specific promoter linked to a gene to effect male sterility, fertility or otherwise express a gene in a specific tissue. However, these patents do not teach the use of this male tissue-preferred regulatory region or the use of this male tissue-preferred regulatory region with an exogenous gene as a method of controlling male sterility.
The present invention is directed to a male tissue specific regulatory region and methods of using the same. Expression of an exogenous gene in a male tissue-preferred manner can mediate male fertility and is useful in many systems such as in hybrid seed production.